Peroxynitrite Modulates Release of Inflammatory Mediators from Guinea Pig Lung Mast Cells Activated by Antigen-Antibody Reaction

Abstract

Background: Peroxynitrite (ONOO^–), the product of the reaction between the superoxide anion (·O₂^–) and nitric oxide (NO), is produced during inflammatory disease and may be a major cytotoxic agent. No reports are available as to whether ONOO^– generates or modulates inflammatory mediator release from activated guinea pig lung mast cells. In this study, we explored the modulatory role of intracellular ONOO^– on inflammatory mediator release (histamine and leukotrienes) from activated mast cells. Methods: Guinea pig lung mast cells were purified by the enzyme digestion, and by using the rough and discontinuous Percoll density gradients. Mast cells were sensitized with IgG1 (anti-ovalbumin) antibody and challenged with ovalbumin (OVA). The intracellular ROS formation was determined by following the oxidative production of 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine 123 (DHR), and anti-nitrotyrosine antibody immunofluorescence. Histamine was assayed using a fluorometric analyzer, leukotrienes by radioimmunoassay, intracellular Ca²⁺ levels by confocal scanning microscopy, and PLA₂ activity using prelabeling of [³H]arachidonic acid. Results: ROS detected by DCFH-DA weakly increased in mast cells activated with OVA (1.0 g/ml), and the ROS so generated was inhibited by ebselen (50 µM). However, the ROS detected by DHR increased 3-fold under the same conditions. Peroxynitrite scavengers sL-MT, DMTU, and inhibitor FeTPPS inhibited ROS formation but the NADPH oxidase inhibitor diphenyleneiodonium (DPI) only partially inhibited this formation. Dimethyl thiourea (DMTU) and seleno-L-methionine (sL-MT) inhibited the tyrosine nitration of cytosolic proteins, the release of histamine and leukotrienes, Ca²⁺ influx, and the PLA₂ activity evoked by mast cell activation. Conclusion: The data obtained suggests that the ROS generated by the antigen/antibody reaction activated mast cells is ONOO^–, and that this modulates the release of inflammatory mediators via Ca²⁺-dependent PLA₂ activity.